Recent evidence suggests that T cells can recognize tumor antigens which probably derive from naturally occurring cellular proteins. These antigens appear to be products of the genetic alterations in the genome that occurs during the process of tumorigenesis. They are therefore analogous to viral antigens and minor histocompatibility (H) antigens, which are processed in the cytosol, and bind to class I major histocompatibility complex (MHC) molecules in the endoplasmic reticulum (ER). The antigen/MHC complex is transported to the cell surface and recognized by cytotoxic T lymphocytes (CTL). The acid-elution method for isolation of naturally processed T-cell epitopes in combination with reverse phase high performance liquid chromatography (RP-HPLC) can provide direct experimental access to these processed peptide sequences. CTL are critical for determining which isolated peptide fragments are relevant antigens. We have demonstrated that tumor-specific CTL can be generated in vivo by genetically altering parental tumor cells to secrete necessary cytokines for T cell activation. The experiments proposed will employ acid extraction and RP-HPLC to identify tumor-specific peptides that are targets of CTL. Specifically, tumors expressing the well-characterized influenza nucleoprotein (NP) gene product will be used to optimize the RPLC-based method for isolating antigenic peptides. This method will then be employed to identify naturally occurring tumor-specific antigens. Isolated peptides will be sequenced and characterized. The efficacy of natural tumor-specific peptides to generate anti-tumor immunity will also be assessed. Once identified, these peptides may lead to substantial advances in our understanding of tumor biology and the anti-tumor immune response. These include: 1) assessment of tumor heterogeneity, 2) elucidation of the process of tumor progression, 3) dissection of the interactions of immune cells, 4) enhancement of the anti-tumor immune response using antigen-derived vaccines, 5) identification of parent proteins that may implicate specific genetic alterations associated with malignant transformation.